The mechanisms by which eukaryotes regulate gene expression are important for understanding many complex biological phenomena including human diseases. Prevention and treatment of such diseases have been and will continue to be improved by basic knowledge of gene regulation, especially because molecular mechanisms of transcriptional initiation are highly conserved in eukaryotic organisms ranging from human to yeast. This proposal will continue to investigate the following basic issues concerning molecular mechanisms of transcriptional regulation, primarily in yeast, by combining chromatin immunoprecipitation (ChIP), molecular genetics, biochemistry, and genomics. 1) Mechanisms of activator-specific recruitment of TFIID and growth-regulation of ribosomal protein (RP) genes: determining functional relationships among proteins associating with RP promoters; defining the activator-TAF interactions in molecular terms. 2) Functional dissection of the Mediator complex: activator-specificity of recruitment to enhancer; Mediator subunits and surfaces necessary for activator-mediated recruitment and connection to the Pol II machinery; addressing whether Mediator truly has a general and required function for Pol II transcription. 3) Genome accessibility to transcription factors: high resolution mapping of nucleosome positions over the entire yeast genome using samples from several yeast species and chromatin reconstituted in vitro; genetic analysis of the mechanism of preferential positioning within coding regions; examining whether principles of nucleosome positioning in yeast extend to human cells. 4) Association of DNA-binding proteins to target sites in vivo: CnlP and microarray technology to examine the effect of protein concentration or presence of activation domains; determining how bZIP proteins with similar DNA-binding specificities in vitro have different target genes in vivo. Evolutionary conservation of binding sites of bZIP proteins in several yeast species and also by human-chimp and human-mouse comparisions. 5) Mechanisms and biological significance of novel genetic elements (ETC loci) containing incomplete Pol III transcription complexes in vivo including examination of a role of the ETC loci in nuclear positioning. 6) Transcriptional elongation using novel in vivo assays: co-association of elongation factors with mRNA coding regions; functional redundancy of elongation factors in terms of Pol II elongation rate and processiyity; role of Swi/Snf nuclepspme-remodeling complex in elongation; initiation vs. elongation as a limiting step for Pol II transcription in vivo; effects of temperature and growth conditions on Pol II transcription. 7) Mechanism by which Hog1 MAP kinase acts as an elongation factor specific for osmotically induced genes: effect of Hog1 on elongation rate and processivity; DNA or RNA sequence [unreadable] [unreadable] [unreadable]